1. Field of the Invention
The present invention relates to an expression vector system for variants of coagulation factor VIII (FVIII) and von Willebrand factor (vWF), more particularly to mutant vWF the size of which is significantly reduced by deleting exons but which has remarkably increased FVIII stabilizing and activating efficiency, and an expression vector system useful for the treatment of hemophilia which is capable of expressing the same along with FVIII.
2. Description of the Related Art
Hemophilia A is a hereditary, X chromosome-linked blood clotting disorder caused by a deficiency in FVIII. Symptoms include frequent bleeding in muscles, bones, digestive and urinary tracts, etc. accompanied by swelling and pain. Current treatment is based on regular supplementation of FVIII. This requires a lifelong treatment, giving troubles in daily lives and economic burdens. Further, there is a high risk of secondary infection during its administration.
FVIII is a large glycoprotein of 180 Kb, and consists of A1-A2-B-A3-C1-C2 domains. The FVIII gene is located on the X chromosome, and its synthesis is carried out mostly in the liver. Until now, there have been a lot of researches to transducer FVIII, but there were many difficulties because its size was too large, or the transduced FVIII gene was not expressed or secreted well. The B domain of FVIII consists of a large exon and its asparagine, serine and threonine residues are highly glycosylated. According to recent functional studies, the domain is not essential in procoagulant activity, and the deletion thereof does not affect the function of FVIII. When B-domain deleted FVIII (BDD-FVIII) was expressed in cells, the problems of unstable FVIII mRNA structure and interaction with ER chaperones were overcome and a lot of FVIII mRNA could be attained. Of the BDD-FVIII, a variant with 226 amino acids at the N-terminal with 6 consensus site for N-linked glycosylation exhibited significantly increased FVIII secretion.
In genetic treatment of hemophilia A, the target cell is bone marrow cells, especially stem cells or progenitor cells. Lentivirus-based vectors are used to transfer the gene. After infection into cells, these vectors insert the gene into the chromosome of the infected cell, thereby enabling stable and consistent expression. Other viruses such as Moloney murine leukemia virus could not be used to infect stem cells or progenitor cells, because they infect only dividing cells. And, although adenovirus produces a large amount of expressed proteins, a consistent expression was impossible because the gene is diluted as the differentiation continues.
Accordingly, a safe and consistent way of transducing FVIII is necessary, and the development thereof is needed. Lentiviral vectors can infect nearly all non-dividing cells, as well as dividing cells, and provide stable expression for a long period of time because they are inserted in the cell chromosome after the infection. Thus, lentivirus-based vectors for expression of FVIII may be useful for gene therapy.
vWF plays an important role in activating FVIII during blood coagulation. vWF is a blood glycoprotein which binds to FVIII thereby preventing it from being degraded in the blood. Besides, it plays a major role in blood coagulation by binding to collagen or platelet when endothelial cells are inured. vWF consists of D′-D3-A1-A2-A3-D4-B1-B2-B3-C1-C2 domains, and the D′-D3 domain binds to FVIII. vWF is a 250 kDa-sized protein and its gene is about 9 Kb in size. Accordingly, it is impossible to insert vWF in a lentiviral vector to help the function of FVIII. Through researches on the essential part in the vWF domains with respect to activation of FVIII, the inventors of the present invention found out that the portion of the vWF gene up to exon 32 functions most efficiently. Based on this finding, we inserted FVIII, an internal ribosome entry site (IRES) and vWF in a lentivirus-based vector. The resultant viral vector expresses the proteins gag-pol, env, tat and rev required for lentivirus, thereby expressing FVIII and vWF upon infection of cells. This attempt has never been made and is valued very highly for gene therapies and hemophilia researches in the future.